Induction of gamma-globin chains which are normally synthesized during fetal life, can improve globin chain balance and ameliorate the clinical outcome and prognosis of β-thalassemia patients. Several compounds capable of inducing fetal hemoglobin, such as short chain fatty acids, aromatic fatty acid and 5-azacytidine, have been so far evaluated in pilot studies. However problems related to the dosing schedule, potency and to the mechanisms of action remain to be solved. In order to make available new compounds for further clinical studies, we evaluated the effect of four oxymethylene diesters provided by Beacon Laboratories on in vitro erythroid progenitor differentiation/maturation and on fetal hemoglobin synthesis. Cultures were set up from peripheral mononuclear cells of normal donors and β-thalassemic patients. The method consisted in a two-phase liquid culture procedure ( 19 days of culture) according to Fibach et al with minor modifications. Mononuclear cells were grown for 7 days (phase I) in alpha-minimal essential medium supplemented with 10% fetal calf serum (PCS) and 5637 conditioned medium plus cyclosporin A. In phase II, the non-adherent cells representing CD-34, BFU-E and CFU-E were recultured in presence of EPO ( 1 U/mL). Erythroid cells differentiation, proliferation and maturation were evaluated at the end of culture (19th day) by cell counts and morphological observation at optical microscope. Cell hemoglobinization was assessed by alkaline benzidine staining. Viability of the cells was established by trypan blue exclusion test. Previous time-course experiments allowed to establish that, in this culture system, the effect on erythroid differentiation is observed by the addition of test materials on the sixth day of phase II (13lh day of culture) To start the study the test compounds were added at different concentrations (100, 250, and 500|xM) at this culture time. At the concentration greater than 100 JlM, three of the test compounds inhibited cell proliferation. The notable exception was BL-1065. At the lowest concentration (100 |lM) BL-1065 increased cell number by three fold compared to untreated control cultures. Additional testing was performed with BL-1065 with concentrations ranging from 5 to 50 (4.M in cultures from normal and thalassemic samples and the effect on proliferation was even more evident. The results of gamma-chain mRNA expression evaluated by RT-PCR and fetal Hb synthesis measured by HPLC will be also presented.
|Issue number||11 PART II|
|Publication status||Published - 2000|
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